i. Field of the Invention
The present invention relates generally to methods for assaying compounds containing primary amino groups and more particularly to a chemiluminescence method wherein a fluorescer analyte containing the primary amino group is chemically excited to yield a readily detectable and quantifiable species.
ii. Description of the Prior Art
Fluorescence detection techniques find wide application in the field of analytical chemistry. Such techniques typically involve the exposure of a sample containing one or more moieties of interest to light of a given wavelength whereby a characteristic emission is observed with respect to each of the moieties. It is then possible to obtain both a qualitative and quantitative assay of the sample.
The problem with such absorption detection techniques, however, is that they often give rise to a high background signal which reduces their sensitivity. More specifically, in fluorescence detection techniques, characterized by light induced emission from the singlet excited state, stray light as well as other scattering phenomena namely, Raleigh and Raman scattering, result in an increase in the background signal thereby reducing the sensitivity of fluorescence detection techniques. Interference from both stray light and Rayleigh scattering can be eliminated by employing properly designed cell compartments and by using emission filters and monochromators. Although interference from Raman scattering can also be eliminated, such requires even more sophisticated techniques. Accordingly, although it is possible to substantially eliminate the background interference in fluorescence analytical techniques, such is achieved only by virtue of a great deal of additional time and expenditure.
Because of the difficulties encountered in the art with respect to high sensitivity fluorescence detection techniques, techniques employing chemiluminescence have been sought. Unlike fluorescence techniques wherein a compound of interest is raised to the singlet excited state by the absorption of ultraviolet or visible radiation, chemiluminescence techniques effect excitation of the compound of interest by a chemical reaction. They therefore do not give rise to the same light scattering phenomena observed with respect to absorption techniques.
To perform chemiluminescence techniques, a chemical reaction is carried out which transfers energy to a fluorescer (or energy acceptor) thus raising the fluorescer to an excited state. The excited fluorescer itself then returns to the ground state by releasing a photon or by other non-emissive processes. The photon release, of course, is registered by a suitable detection device.
Despite the indisputable advantages of chemiluminescence techniques over fluorescence techniques, the practice of chemiluminescence techniques has not been widespread. The reason for this relates to the extreme difficulty with which compounds can be made to exhibit chemiluminescence. More specifically, the number of compounds which exhibit fluorescence, i.e., which emit a photon upon excitation by light, is quite limited to begin with. Of this limited number of fluorescence compounds, only a still smaller number of compounds will exhibit chemiluminescence, i.e., emit a photon upon excitation by a chemical reaction. Add to this the fact that application of chemiluminescence to assaying techniques requires that the compound exhibiting chemiluminescence be derivable from the materials of interest (primary amino compounds in the case of the present invention) and it is not surprising that there are few effective assaying methods of employing chemiluminescence techniques which are available.
In one chemiluminescence technique, a mixture of an aryl oxalate with hydrogen peroxide is used to activate a fluorescer formed from dansylated amino acids, i.e., amino acids reacted with 5-dimethylaminonaphthalene-sulfonyl chloride followed by acid hydrolysis. The individual dansylated amino acids themselves are initially fractionated by high performance liquid chromatography. The detection limits for such activated amino acid fluorescers is 2 to 5 femtomoles when detection is carried out with a specifically designed detector employing a spiral flow cell interfaced to a single photon counting photomultiplier. Fluorescers formed by dansylating amino acids and primary amines were shown to give the lowest detection limits, i.e., 0.8 to 14 femtomole for various aliphatic primary amines. Fluorescers formed by reacting aliphatic amines with 4-chloro-7-nitrobenzo-1,2,5-oxadiazole (NBD-Cl) and o-phthaldehyde (OPA) gave detection limits ranging from 19 to 270 and 94 to 580 femtomoles respectively.
Although the above reported detection limits obtainable by employing dansylated derivatives as fluorescers in chemiluminescence assaying techniques are quite good, their applicability to samples which are fractionated by high performance liquid chromatography techniques is somewhat limited. Reverse phase high performance liquid chromatography techniques, i.e., wherein the mobile phase is organic-water mixture typically require the presence of substantially and highly variable concentrations of water. The fluorescence yields .phi.f of dansylated amino acids, however, are highly affected by such variations in solvent. Thus, dansylated amino acids which have .phi.f values of 0.3 to 0.7 in organic solvents were found to have .phi.f values of 0.053 to 0.091 in water. Accordingly, the fluorescence yield .phi. of dansylated amino acids fractionated by high performance liquid chromatography is often at an unacceptably low level for detecting and quantifying very low amounts of those amino acids.